1. Field of the Invention
This invention relates to methods useful in determining the nucleotide sequence of nucleic acids. More specifically, it relates to nucleic acid sequencing methods in which the nucleic acid acts as a template for the production of a complementary polymer by a polymerase enzyme and in which the incorporation of a nucleotide into the growing end of the lengthening complementary nucleic acid polymer is detected.
2. Description of Related Art
DNA sequencing has become an essential tool in molecular genetic analysis and is now commonly used to sequence large genomes of higher organisms, short regions of DNA to identify mutations of interest, single nucleotide polymorphisms (SNPs), and complete genes and associated upstream and downstream control regions. New techniques are needed that are particularly suited for short or medium-long DNA sequencing projects such as SNP genotyping (Ahmadian, Gharizadeh et al. 2000). Faster, less expensive and easy to implement sequencing methods will lead to improved SNP discovery. Now that the human genome has been sequenced, there is a need for rapid and inexpensive methods with improved read length and throughput that can be used to correlate phenotype and genotype differences for personalized genomics, personalized medicine, and the routine study of individual genomes (Chan, 2005).
Numerous methods can be used to sequence DNA. Wu and Taylor used methodology derived from RNA sequencing to sequence the cohesive ends of Phage λ DNA (Wu and Taylor, 1971). The method was not, however, easily applied to large-scale DNA sequencing. Plus minus sequencing was developed and used to sequence the phage ΦX174 genome (Sanger and Coulson, 1975) but was improved by Sanger's chain termination method (Sanger et al., 1977). In the chain termination method, 2′,3′-dideoxynucleoside triphosphates act as specific chain-terminating inhibitors of DNA polymerase. The method produces a series of DNA fragments of different lengths. DNA fragments of different lengths can also be produced using chemical agents to produce DNA fragments by breaking the DNA molecule at adenine, guanine, cystosine, or thymine (Maxam and Gilbert, 1977). Initially, the Klenow fragment of DNA polymerase was used because this fragment does not have exonuclease activity. Li demonstrated greater accuracy and longer read lengths with native Taq DNA polymerase (Li et al., 1999). In the Sanger method, DNA synthesis is carried out in the presence of the four deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) in four separate reaction mixes containing a low concentration of one each of the four dideoxynucleoside triphosphate analogues. One or more of the dideoxynucleoside triphosphates is labeled. To determine the nucleotide sequence, the DNA fragments are denatured and separated by high resolution gel electrophoresis and the order of the four bases can be read from the gel by measuring the location of each fragment in the gel by detecting the label using radioactivity or fluorescence. Methods to prepare DNA for sequencing have been automated (Lidstrom and Meldrum, 2003) as have the sequencing methods described above (Lidstrom and Meldrum, 2003) but there are still major disadvantages to their use including the cost of the required equipment, the need for highly trained technicians, the laborious nature of the methods, the time required for sequencing, the requirement for labeling, extensive space requirements, and the fact that often more information is generated than is needed. Additionally, high accuracy requires several-fold coverage of a genome (Chan, 2005).
If the objective of sequencing is to identify mutations or SNPs, short sequences of DNA are often analyzed from many different samples. Sanger sequencing can be used for this purpose but is expensive, time consuming, and generates more data than is needed. Current methods for sequencing short segments of DNA include sequencing by hybridization, Pyrosequencing, and massively parallel signature sequencing (MPSS).
Pyrosequencing determines the DNA sequence by identifying which of the four bases is incorporated at each step in the copying of a DNA template by DNA polymerase without the need for electrophoresis, radioactivity, or fluorescence (Hyman, 1988). In this method, the DNA segment to be sequenced acts as a template for the production of a complementary DNA segment by a DNA polymerase. As DNA polymerase moves along the single stranded DNA template, each of the four nucleoside triphosphates is made available sequentially and, following adequate reaction time, is removed. Pyrophosphate (PPi) is released when one of the four bases is incorporated into the growing nucleic acid polymer by DNA polymerase. A number of methods can be used to detect the released pyrophosphate including the enzymatic method originally proposed by Hyman (Nyren and Lundin, 1985) in which ATP sulfurylase and luciferin are used to produce a light emission proportional in intensity to the amount of PPi as shown in the following reactions:

Nyren et al. showed that the sequencing method proposed by Hyman could be accomplished in the solid phase on immobilized DNA fragments (Nyren et al., 1993) and others have shown that the method may be used on double-stranded DNA (Nordstrom et al., 2000). The method as originally proposed by Hyman suffers from a number of drawbacks. Most notable is the fact that luciferase is not entirely specific for ATP as a high energy substrate and can also react with deoxyadenosine-5′ triphosphate and to a lesser extent may react with other nucleoside triphosphates. Additionally, PPi is a contaminant of some commercially sold chemicals and is difficult to remove from buffers. Small nuclease contaminants in the buffers can potentially catalyze the formation of PPi via the reaction dNTP→dNMP+PPi.
To improve the signal to noise ratio of the Hyman method, Ronaghi et al. substituted deoxyadeno sine α-thiotriphosphate (dATPαS) for the natural deoxyadeno sine triphosphate (Ronaghi et al., 1996). They showed that dATPαS is incorporated into a growing strand of DNA polymer by DNA polymerase but is not recognized by luciferase. A further refinement of the Hyman method eliminated the need for intermediate washing steps needed to remove unincorporated nucleotides by the addition of a nucleotide-degrading enzyme to obtain a four-enzyme mixture, DNA polymerase, ATP sulfurylase, firefly luciferase and a nucleotide-degrading enzyme such as apyrase (Ronaghi et al., 1998). Despite this improvement, an inherent problem with the method is the difficulty in determining the number of incorporated nucleotides in homopolymeric regions due to the nonlinear light response following incorporation of more than three or four identical nucleotides (Ronaghi et al., 1998).
In view of these and other deficiencies in the art, a need exists for more accurate and less-expensive methods of sequencing DNA and other nucleic acid polymers to be developed.